PROJECT SUMMARY/ABSTRACT During mammalian embryogenesis, carefully orchestrated regulation of gene expression guides the differentiation of multiple cell types from a single genome. Great strides have been made to understand gene regulation, in particular, the development of methods to detect protein-DNA interactions such as chromatin immunoprecipitation assays and related techniques. The application of these techniques to the study of early mammalian embryos, however, is hampered by the small size of the conceptus and the need to study subsets of cells within embryos. Moreover, the temporal and spatial features of the protein-DNA interactions, essential to understand pattern formation, are lost because of the need to homogenize tissues. In this proposal we aim to generate a novel approach to visualize protein-DNA interactions in their natural setting in developing embryos. The strategy combines recently developed CRISPR/Cas9 methodology, proximity ligation assays and viral transduction techniques. Our approach offers significant improvements over currently available methods: It minimizes the amount of tissue required to detect protein-DNA interactions, allows the spatial localization of protein-DNA interactions, provides single cell resolution and offers the possibility of assessing the evolution of gene regulatory interactions over time. In essence, the equivalent of chromatin immunoprecipitation assays with single-cell resolution in a wholemount format. The basal nature of the proposed research has the potential to significantly impact several fields of research, that include gene regulation studies, mammalian developmental biology and stem cell research. These in turn are basic for understanding normal and abnormal human development and the development of stem cell technology applicable in regenerative medicine.